The expression of molecular chaperones has been shown to suppress protein misfolding/aggregation and cellular toxicity phenotypes in model systems associated with Huntington's Disease, Alzheimer's Disease, Parkinson's Disease, and ALS. A feature common to diseases of protein conformation is the appearance of folded intermediates that self-associate to form protein aggregates and inclusions. The molecular chaperones Hsp90 and Hsp70 sequester damaged proteins that appear in cells exposed to physiological and environmental stress. The ability of molecular chaperones to suppress the cellular toxicities associated with expression of these "toxic" proteins may be due to the intrinsic properties of chaperones to capture and suppress the appearance of folded intermediates. Therefore, we propose that the identification of small molecules that elevate the expression of genes encoding heat shock proteins and molecular chaperones should lead to the development of novel therapies beneficial to the prevention of neurodegenerative diseases. The rationale for this proposal is based on results obtained by our laboratory and others who participated recently in a screening program organized by the NINDS, Huntington Disease Society of America, Hereditary Disease Foundation, and the ALSA to identify new drugs for treating these diseases. A search was carried out for drugs that activate the heat shock response; the most effective compound identified was the natural product celastrol. Synthetic analogs of celastrol will be prepared to optimize its effectiveness as a regulator of the heat shock response and a suppressor of neurotoxicity and to determine its mechanism of action as an activator of the heat shock response. To probe the function of celastrol as a potential therapy for neurodegenerative diseases, the following Specific Aims will be addressed: (1) Synthesize analogs of celastrol that induce the human heat shock response using a heat shock promoter-reporter assay in human tissue culture cells. (2) Determine the mechanism of action of celastrol (or an analog). The working model is that celastrol activates the heat shock response by inducing heat shock transcription factor HSF1. The mechanism by which HSF1 activity is induced by celastrol will be determined. It also will be determined whether celastrol, by virtue of its ability to activate the expression of chaperones, can reduce the aggregation and neurotoxicity of the Huntington Q64 protein expressed in a human SH-SY5Y neuroblastoma cell line. (3) Studies will be carried out to identify the binding target for celastrol using molecular biological and biochemical techniques. Identified target(s) will then be cloned and characterized. Results of these studies will aid in the determination of the molecular mechanism of celastrol action and in the further understanding of the heat shock response, identify new targets for therapeutic intervention, and may also lead to new therapies for neurodegeneration.